Doctor of Philosophy / Department of Chemical Engineering / Peter H. Pfromm / The expanded Renewable Fuel Standard (RFS2), established under the Energy Independence and Security Act (EISA) of 2007, mandates the production of 136.3 GL/year of renewable fuels in the U.S. in 2022: 56.8 GL/year of corn-ethanol, 60.6 GL/year of second generation biofuels such as cellulosic ethanol, and 18.9 GL/year of advanced biofuels such as biomass-based diesel. One of the several challenges when a biochemical conversion technique is used to produce bioethanol from corn and cellulosic feedstock is the high energy demand for recovering and purifying ethanol, which is mainly due to the low concentration of ethanol in the fermentation broth and the challenging water-ethanol vapor liquid equilibrium. Dilute ethanol from the fermentation broth can be separated and concentrated aided by salt extractive distillation to directly produce fuel ethanol leading to significant energy savings. Techniques other than highly energy intensive evaporative salt concentration/crystallization and solids drying for recovering salt, which is used to facilitate distillation, have rarely been considered. In this study, a novel combination of electrodialysis and spray drying was investigated to recover the salt. Salt extractive distillation – with salt recovery enabled by electrodialysis – was conceptually integrated in the fermentation broth-ethanol separation trains of corn and cellulosic ethanol facilities and investigated through process simulation with Aspen Plus® 2006.5 to reduce the recovery and purification energy demand of bioethanol. Experiments for the electrodialytic concentration of calcium chloride from high diluate concentrations, prevalent in the salt recovery process when calcium chloride is used as the salt separating agent in the salt extractive distillation of bioethanol, were carried out to determine the fundamental transport properties of an ion exchange membrane pair comprising commercially available membranes for implementation in the conceptual process designs. The maximum calcium chloride concentration achievable through electrodialytic concentration is 34.6 wt%, which is mainly limited by the water transport number. In case of corn-ethanol, retrofitted salt extractive distillation resulted in an energy demand reduction of about 20% and total annual cost savings on the order of MM$0.5 per year when compared with the state-of-the-art rectification/adsorption process for producing fuel ethanol from the beer column distillate. In case of cellulosic ethanol, salt extractive distillation with direct vapor recompression provided the highest energy savings of about 22% and total annual cost savings on the order of MM$2.4 per year when compared with the base case comprising conventional distillation and adsorption for recovering and purifying ethanol from the fermentation broth. Based on the conceptual process design studies, an overall maximum energy savings potential of 1.5*10[superscript]17 J or about 0.14 Quad (as natural gas higher heating value) per year could be estimated for the targeted 56.8 GL of corn-ethanol and 60.6 GL of cellulosic ethanol to be produced in the U.S in 2022 when salt extractive distillation enabled by electrodialysis is implemented in the fermentation broth-ethanol separation trains of the corn and cellulosic ethanol facilities.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Donghai Wang / Lignocellulosic biomass such as agricultural residues tends to be a sustainable feedstock for production of biofuels and biobased products in the long term due to its high availability and relative low cost. However, conversion of lignocellulosic biomass to biofuels faces significant technical challenges. One of the major challenges is biomass logistics. The agricultural residues are often harvested during a limited harvest season and stored as bales with low bulk density, making them difficult to handle, transport, store, and use in their natural forms. Densification of biomass by pelleting process could significantly improve the bulk density of biomass and thus improve handling efficiency and reduce transportation and handing costs. The main focus of this research was to better understand the impacts of pelleting process as well as pelleting conditions on physical properties, chemical compositions, biomass structure, and fermentable sugar yield of sorghum stalk, corn stover, wheat straw, and big bluestem. Results showed that pelleting process can increase biomass density up to 9-12 folds. Pelleting condition such as hammer mill screen size and ring-die pelleting mill die thickness had significant effects on bulk density, true density, and durability of biomass pellets. Although the pelleting process did not show significant effects on chemical composition of biomass before dilute-acid pretreatment process, glucan content of biomass pellets increased with the increase in ring-die pelleting mill die thickness and decreased with the increase in mill screen size after dilute-acid pretreatment. Opposite trend was observed for xylan content of biomass pellets as affected by pelleting conditions after dilute-acid pretreatment process. Biomass crystallinity increased after pelleting process, but not in a significant level. Softened surface region of biomass was removed after pelleting process, making the biomass more amendable to enzymatic attack. In this study, the optimum pelleting conditions were to grind the biomass feed using a 6.5-mm mill screen and to pellet biomass using a 44.5-mm ring-die pelleting mill die thickness. Under this optimum pelleting condition, the enzymatic conversion of cellulose of wheat straw pellets was the highest (94.1%), followed by corn stover pellet (93.1%), sorghum stalk pellet (92.1%), and big bluestem pellet (91.1%).

Idag produceras bioetanol från grödor som sockerrör, majs, vete osv. Det är ett etiskt dilemma att använda potentiell mat för produktion av bränsle när det samtidigt som det råder matbrist i många länder. En bättre lösning är att använda restprodukter istället. I projektet utnyttjades restprodukter som bröd.  Bröd innehåller stärkelse som består av glukosmonomerer. Idag finns många intressanta produkter som skulle kunna utnyttjas istället för att använda matprodukter som nämndes ovan. Två olika brödsorter användes i experimenten som kommer från lantmännen. De två olika brödsorterna var korvbröd och brödmix (blandade brödrester). Anledningen till varför olika sorter av bröd användes var för att kunna jämföra om det finns någon skillnad mellan produkten. För att kunna se det, behövdes bröd förberedas och med samma faktorer som temperatur, koncentration och alla steget som den måste gå igenom dvs förvätskning, sackarifering och fermentering.  För kunna produceras etanol från brödrester behövdes två steg av stärkelsehydrolys. Första steget kallas förvätskning och med hjälp av enzymet alfa-amylas kunde stärkelsens kedja klippas till kortare kedjor. Viskositet testades efter förvätskning för att kunna bestämma vilken eller vilka koncentrationer skulle process kunna utföras. Det visades sig att mellan 10 och 30 procent av bröd går att använda. Efter detta steg skedes en annan process som kallas sackarifiering som utfördes med enzymet gluko-amalys för att frigöra glukos. Detta steg användes framför allt för när jäst användes. Jäst har inte förmåga att produceras eller omvandlas kolhydraten till glukos.  Svamp- och jäst-mikroorganismer användes i processen. Efter sackarifiering tillfördes mikroorganismerna i processen som kallas jäsning eller fermentering för att producera etanol och djurfoder som är slutprodukter. I projektet användes olika former av mikroorganismer som pellet, filamentös och sporer som inokulat. Detta gjordes för att se om det finns någon skillnad mellan olika formerna.  Det spelade ingen roll vilken typ av bröd som användes. Högsta koncentration av etanol var ca 26 g/L.  I projektet utnyttjades även tunndrank som kommer också från lantmännen. Anledningen var att minska användnings av rent vatten vid etanolproduktion. / Today bioethanol is produced from crops such as sugar cane, corn, and wheat. It is an ethical dilemma to use potential food for fuel production when a lot of countries do not have enough food. There is a better solution for this case and that is to use waste products. In this project exploitation of bread waste was investigated. Bread is rich in starch which consist of glucose monomers. Two types of bread waste were used in the experiment: hotdog bread and mixed bread. The reason why two types of breads were used was to compare if there are any differences between the products. The bread was prepared via liquefaction and saccharification using different factors like temperature and solid concentration, and was then fermented.   In the first step, liquefication, alpha-amylase was used to cut the starch polymer to shorter oligomers. The viscosity was tested after liquefaction to determine which bread concentrations could be used. It turned out that between 10 and 30 percent of bread was usable. In the next step, saccharification, gluco-amylase was used to release glucose monomers. This step was used mainly when yeast was used.  Both fungi and yeast-microorganisms were used in the process. After saccharification the microorganisms were added to the process to carry out fermentation to produce ethanol and animal feed as final products. In the project different forms of fungi were used for inoculation like pellets, filamentous, and spores. This was done to see if there were any difference between the forms.   The type of bread that was used did not matter. The highest concentration of ethanol was ca 26 g/L.  In the project thin stillage from was also used. The reason was to reduce the amount of clean water for ethanol production.

O presente trabalho teve por objetivo avaliar condições de imobilização da levedura Scheffersomyces stipitis NRRL Y-7124 pelo método do aprisionamento em gel de alginato de cálcio visando à produção de bioetanol em biorreator STR tipo cesta à partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Primeiramente, realizou-se as etapas de obtenção, destoxificação e caracterização do hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Realizou-se em seguida um screening objetivando a seleção de um meio de cultivo adequado para a produção de etanol por esta levedura. O meio escolhido foi aquele onde se suplementou o hidrolisado com extrato de levedura (3,0 g/L), peptona (5,0 g/L), (NH4)2SO4 (2,0 g/L) e CaCl2 (0,1 g/L), onde verificou-se um fator de conversão de xilose à etanol (Yp/s) de 0,33 g/g. As condições de imobilização da levedura foram então avaliadas por planejamento fatorial 23 completo onde os fatores concentração de alginato de sódio, concentração do cloreto de cálcio e tempo de cura foram investigados. Após a análise estatística, as condições 2% de alginato de sódio, 0,1M de cloreto de cálcio e tempo de cura de 12 horas foram fixadas para as etapas seguintes. Nestas condições, avaliou-se então a influência da concentração de células à serem imobilizadas e agitação durante a fermentação a partir de um planejamento fatorial 22 completo, definindo-se assim 10 g/L de células e 100 rpm como condições ideais. Após a determinação das condições de imobilização do processo, verificou-se a estabilidade das células imobilizadas em repetidos ciclos fermentativos, para isso cinco bateladas repetidas em frascos Erlenmeyer foram realizadas. Observou-se que apesar da levedura assimilar xilose e produzir etanol em todos os ensaios, uma diminuição na eficiência da fermentação foi verificada, diminuindo em 24% da terceira para a quarta batelada, indicando assim que a levedura imobilizada era viável para o sistema de batelada repetida em até 3 ciclos nas condições estudadas. Iniciou-se então ensaios fermentativos em biorreator STR tipo cesta, realizando-se ensaios em meio sintético e em hidrolisado hemicelulósico. Observou-se reprodutibilidade nos ensaios utilizando os diferentes meios, com um valor de Yp/s de 0,21g/g e uma produtividade volumétrica de 0,15 g/L.h em ambos os ensaios. Fermentações em sistema de bateladas repetidas foram realizadas neste biorreator STR tipo cesta. Realizou-se cinco ciclos consecutivos, ao final dos quais observou-se comportamento semelhante às bateladas repetidas realizadas em frascos Erlenmeyer, na qual a partir de três ciclos a capacidade fermentativa da levedura S. stipitis diminuiu, apresentando uma produtividade volumétrica em torno de 0,16 g/L.h nas três primeiras bateladas. O gel de alginato de cálcio apresentou considerável estabilidade em sistema de bateladas repetidas indicando a possibilidade de sua utilização nesse processo. Embora os resultados obtidos neste trabalho sejam inferiores aos observados com células livres por outros autores, os mesmos demonstraram o potencial do emprego do gel de alginato de cálcio e da levedura Scheffersomyces stipitis imobilizada para a produção de bioetanol a partir de bagaço de cana-de-açúcar e contribuíram para os conhecimentos sobre a fermentação de hidrolisado hemicelulósico à etanol. / This study aimed to evaluate immobilization conditions for the yeast Scheffersomyces stipitis NRRL Y-7124 entrapped in calcium alginate gel in basket type of STR bioreactor for ethanol production from sugarcane bagasse hemicellulosic hydrolysate. For this purpose, first the steps to obtain the hydrolysate by dilute acid pretreatment, detoxification and characterization of hydrolysate was performed. Then, a screening aiming the selection of a suitable culture medium suitable for ethanol production by this yeast was carried out. The medium which showed maximum ethanol production (Yp/s, 0.33 g/g) was selected to continue the further studies. It was composed by the hydrolyzate supplemented with yeast extract (3.0 g/L), peptone (5.0 g/L), (NH4)2SO4 (2.0 g/L), CaCl2 (0.1g/L). The immobilization conditions of the yeast were then evaluated through a 23 factorial design where the three process variables i.e. concentration of sodium alginate, concentration of calcium chloride and reaction time were investigated. After statistical analysis, the optimum set of conditions (2% of sodium alginate, 0.1 M of calcium chloride and a reaction time of 12 hrs) were set to perform the following steps of this study. Subsequently, the influence of the cell concentration for immobilization and agitation during fermentation were studied considering a factorial design 22. This study revealed that 10 g/L of cells and 100 rpm were the optimum conditions for ethanol production via immobilized systems. After determination of the conditions for immobilization procedure, the stability of the immobilized cells were evaluated by repeated fermentation cycles, for that five repeated batches were performed in Erlenmeyer flasks. It was observed that despite the yeast assimilates xylose and produces ethanol in all assays, a decrease in the efficiency of the fermentation was verified from the third batch, revealing the 65% efficiency in the second batch and 39% in the fourth batch. This behavior indicates that the immobilized yeast is viable for repeated batch system only up to 3 cycles under the employed conditions. Fermentation tests in basket type STR bioreactor were carried out using synthetic medium and hemicellulosic hydrolysate as carbon source. Reproducibility was observed in assays using the different medium with ethanol yield (Yp/s) of 0.21 g/g and a volumetric productivity of 0.15 g/L.h in both assays. Fermentation assay in repeated batch system were carried out in STR basket type bioreactor. Five consecutive fermentation cycles were performed which eventually showed the similar behavior with the repeated batches conducted in Erlenmeyer flasks. The fermentative efficiency of the yeast S. stipitis was considerably good up to three cycles with a volumetric productivity of 0.16 g/L.h followed by a concomitant down fall. The calcium alginate gel showed a considerable stability in the experiments, indicating the viability of its application in repeated batch system. Although the results of this work are inferior to that observed by other authors using free cells, the calcium alginate gel potential is evident and the yeast Scheffersomyces stipitis showed to be capable to produce ethanol in immobilized form, contributing with knowledge for second generation ethanol production from sugarcane bagasse hemicellulosic hydrolysate adopting biochemical platform.

Resíduos celulósicos por erem ricos em celulose e hemicelulose estão sendo apontados como promissoras fontes para a produção de etanol. Com isso, este trabalho tem como objetivo hidrolisar e fermentar resíduos celulósicos visando a produção de etanol. Para estudo da hidrólise do bagaço de cana-de-açúcar, foram isolados 22 fungos da cidade de Araras-SP e estes testados para produção de endoglucanases (EG). Foram testadas temperatura e pH ótimos, diferentes tampões, estabilidade do coquetel e influência de sais, EDTA e ?-mercaptoetanol para lacase, xilanase, endo-?-1,4-glucanase, celobiohidrolase I, ?-glucosidase e glucoamilase. As condições padronizadas para aplicação do coquetel enzimático foi 55ºC, tampão citrato de sódio 50 mM e pH 5,0. Para elaboração do coquetel enzimático foi realizado um Plackett-Burman seguido de um delineamento composto central rotacional, em que as condições padronizadas para aplicação foram (U/g de bagaço de cana-deaçúcar): 0,122 de lacase; 7 de xilanase; 5 de endoglucanase; 14 de celobiohidrolase e 9 de ?-glucosidase. Foram liberados 4,405 µmol de açúcares redutores por mL, após 48 horas de hidrólise do bagaço a 55ºC, em tampão citrato de sódio 50 mM (7 mL), pH 5,0, agitação de 110 rpm. O hidrolisado obtido foi fermentado pelas leveduras Saccharomyces cerevisiae PE-2 e Meyerozyma guilliermondii 311 (CCT7783), separadamente e em cultivo misto, averiguando a necessidade de suplementação do meio, para a produção de etanol. Os resultados mostraram a necessidade de suplementação para aumento da viabilidade das leveduras. Observou-se que a suplementação manteve a viabilidade acima de 90%. A fermentação com Meyerozyma guilliermondii 311 (CCT7783), sem a necessidade de suplementação, possibilitou a produção de 12,66% de etanol; com a Saccharomyces cerevisiae a produção de etanol foi de 7,03%, quando o meio foi suplementado com 3 g/L de extrato de levedura. A fermentação com cultura mista produziu 0,53% e 1,18% de etanol em 24 e 48 horas de cultivo, respectivamente. Para aplicação enzimática na hidrólise do lodo branco foram utilizadas amilase de Aspergillus carbonarius e celulase comercial. Os produtos de hidrólise enzimática do lodo branco detectados HPLC mostraram a formação de glicose, xilose e traços de maltose. A viabilidade celular da levedura Saccharomyces cerevisiae durante a fermentação se manteve bastante elevada, acima de 95 %. A fermentação do lodo branco, quando o meio foi suplementado com 3g/L de extrato de levedura, foi considerada satisfatória, com 2,37 g/L de etanol. Foram realizadas análise de superfície por ion-tof e microscopia eletrônica de varredura nos resíduos celulósicos. Desta maneira, com este trabalho foi possível estudar a importância da aplicação de enzimas fúngicas para degradação de biomassa lignocelulósica para produção de etanol de segunda geração. / As cellulosic wastes are rich in cellulose and hemicellulose they have been pointed as promising sources for ethanol production. Thereby, this work aimed to hydrolyze and ferment cellulosic residues to produce ethanol. In order to study the hydrolyzes of sugar-cane bagasse and ferment the sugars obtained from this process, twenty-two fungi were isolated in the city of Araras-SP and tested for endoglucanase (EG) production. Aiming to optmize an enzyme cocktail, variables as temperature, optimum pH, different buffers, stability, salt influence, EDTA and ?-mercaptoethanol were tested for laccase, xylanase, endo- ?-1,4-glucanase, cellobiohydrolase I, ?-glucosidase and glucoamylase. The standard conditions for applying the enzyme cocktail were 55°C, sodium citrate buffer 50 mM pH 5.0. For the preparation of the enzyme cocktail a Plackett-Burman was made followed by a central composite design, in which the standard conditions for application were (U/g of sugarcane bagasse): 0.122 of laccase; 7 of xylanase; 5 of endoglucanase; 14 of cellobiohydrolase and 9 of ?-glucosidase per gram of sugarcane bagasse. After 48 hours of hydrolyzes in the following conditions, 55ºC, sodium citrate buffer 50 mM (7 mL), pH 7.0, 110 rpm, 4.405 µmol of reducing sugar per mL were released. The hydrolyzate obtained was separately fermented by the yeasts Saccharomyces cerevisiae PE-2, Meyerozyma guilliermondii 311 (CCT7783) and with both yeasts in a mixed cultivation, in order to investigate the need of medium supplementation for ethanol production. The results showed the need of supplementation to increase the viability of the yeasts. It was observed that this procedure kept the viability over 90%. The fermentation with Meyerozyma guilliermondii 311 (CCT7783), without supplementation, allowed the production of 12.66% of ethanol and with Saccharomyces cerevisiae the production was 7.03% with a supplementation of 3 g/L of yeast extract. The fermentation with mixed cultivation produced 0.53% and 1.18% in 24 and 48 hours of cultivation, respectively. Another objective of this work was the hydrolysis of paper sludge which was obtained by using amylases from Aspergillus carbonarius and commercial cellulases. The products of enzymatic hydrolysis of the white sludge detected in HPLC showed the formation of glucose, xylose and maltose traits. Cell viability counting of the yeast, analyzes of pH, reducing sugar and alcohol content were carried out. Cellular viability of Saccharomyces cerevisiae along the fermentation was kept over 95%. Paper sludge fermentation using Saccharomyces cerevisiae, in medium supplemented with yeast extract 3 g/L, was considered satisfactory, showing 2.37 g/L of ethanol. Surface analysis was performed by Ion-tof and scanning electron microscopy in the cellulosic waste. Thus, this work made possible to study the importance of fungal enzymes application in lignocellulosic biomass degradation for the production of second generation ethanol.

Au cours des dernières années, la synthèse ciblée du butadiène en utilisant le bioéthanol a suscité une attention sans précédent en raison de l'intérêt croissant aux matières premières biosourcées ainsi que de la demande croissante en butadiène.Un processus pertinent dans ce contexte est le processus d'Ostromyslensky, qui s’effectue en deux étapes, comprenant la déshydrogénation de l'éthanol en acétaldéhyde en une étape séparée , suivie de la production de butadiène dans la deuxième étape par réaction d'acétaldéhyde avec de l'éthanol supplémentaire. Bien que la viabilité économique et la faisabilité de ce procédé d’éthanol en butadiène (ETB) soient bien établies, il reste de la place pour de meilleures performances catalytiques et une meilleure sélectivité. Dans cet effort, notre objectif était de développer une famille de catalyseurs sur silice à base de silice bien définis, basés sur la chimie organométallique de surface (SOMC) et de les tester lors de la conversion d'EtOH / AA en BD. Le premier ensemble de pré-catalyseurs a été synthétisé en traitant le [(=SiO)2TaHx] précédemment connu avec du N2O en tant qu'oxydant doux. La deuxième famille de catalyseurs a été préparée par calcination de l'espèce alkyl de tantale à 500°C. Les pré-catalyseurs ont été caractérisés par FTIR, RMN SS, UV-vis-DRS, DRX, EXAFS et HR-STEM. On a découvert que les pré-catalyseurs SOMC oxydés au N2O avaient principalement isolé des espèces [(SiO)2Ta (OH)x] peuplant la surface, tandis que la famille des pré-catalyseurs synthétisés par calcination mettait en évidence un mélange d’espèces de surface, y compris des agrégations de type cordes. Les tests catalytiques sur ces catalyseurs ont donné des résultats prometteurs, présentant une catalyse supérieure dans la transformation d'EtOH / AA en BD en termes de sélectivité en BD et de rendement par rapport à l'état de la technique. Outre l'excellente sélectivité, une gamme étroite de distribution du produit et une formation négligeable de coke ont été observées. Les espèces de TaOx isolées sur le pré-catalyseur oxydé au N2O ont montré une activité nettement meilleure et se sont révélées être les sites actifs de cette conversion par rapport à l'agrégation en chaîne de centres de tantale sur le matériau calciné. Sur la base de ces études DRIFT et in situ sur les catalyseurs, un mécanisme préliminaire pour cette conversion a été proposé / In the recent years on-purpose synthesis of butadiene using bioethanol has gained unprecedented attention owing to rise in interest for bio-based feedstock along with the steeply increasing demand for butadiene (BD). In this regard a relevant process is the Ostromyslensky’s two-step process, involving dehydrogenation of ethanol to acetaldehyde in a separate step, followed by butadiene production in the second stage by co-feeding ethanol and acetaldehyde. Although the economic viability and feasibility of this ethanol to butadiene (ETB) process is well established, there is a room for better catalytic performances and selectivity. In this endeavour our aim was to develop a family of well-defined Ta-based silica-supported catalysts through Surface Organometallic Chemistry (SOMC) and test them in the conversion of EtOH/AA to BD. The first set of pre-catalysts was synthesized by treating the previously known [(=SiO)2TaHx] with N2O as mild oxidant. The second family of catalysts was prepared by calcination of the tantalum alkyl species at 500°C. The pre-catalysts were characterized by FTIR, SS NMR, UV-vis-DRS, XRD, EXAFS and HR-STEM. The N2O oxidized SOMC pre-catalysts were found to have mostly isolated [(=SiO)2Ta(OH)x] species populating the surface whereas the family of pre-catalysts synthesized via calcination evidenced a mixture of surface species, including string-like aggregations.Catalytic tests over these catalysts generated promising results exhibiting superior catalysis in the transformation of EtOH/AA to BD in terms of both BD selectivity and yield compared to the state of the art. In addition to the excellent selectivity a narrow range of product distribution and negligible coke formation was observed. Isolated TaOx species on the N2O oxidized pre-catalyst showed markedly better activity and were found to be the active sites in this conversion compared to the string-like aggregation of tantalum centres on the calcined material. Based on this and in-situ DRIFT studies over the catalysts a preliminary mechanism for this conversion was proposed

Accurate modeling of mixed solvent electrolyte systems is difficult and is not readily available in property modeling software such as Aspen Plus. Support for modeling these systems requires the knowledge and input of parameters specific to the compounds in question. The need for these parameters is particularly relevant in simulating new designs based upon recent developments in a concept known as pass-through distillation (PTD). In support of a specific application of PTD, this work determines and validates with existing experimental data, accurate user-parameters for the eNRTL property model in the ternary system of ethanol, water, and lithium bromide. Furthermore, this work creates the foundation for simulating this new PTD process by modeling the removal of bioethanol from a fermentation broth using low temperature evaporation in conjunction with absorption and stripping units to omit the need of a condenser requiring refrigeration. This will enable future investigations into the applications of PTD as well as provide a foundation for modeling the ternary system of ethanol, water and lithium bromide.

In the future the demand for ethanol is expected to increase greatly due to the rising energy requirements in the world. Lignocellulosic materials are a suitable and potentially cheap feedstock for sustainable production of fuel ethanol, since vast quantities of agricultural and forest residues are available in many countries. However, there are several problems involved in the utilization of lignocellulosic raw materials as sugar source. The most common way of releasing the simple sugars in the material is by dilute acid hydrolysis. This procedure is relatively simple and cheap, but in addition to the sugars it creates inhibitory compounds. These inhibitors make it very hard for the yeast to ferment the hydrolyzate and detoxification is often necessary. One way to overcome this problem is to encapsulate the yeast. Encapsulation is an attractive method since it improves the cells stability and inhibitor tolerance, increases the biomass amount inside the reactor, and decreases the cost of cell recovery, recycling and downstream processing. However, the method does not yet permit long-term cultivation since the capsules used so far are not robust enough. Therefore more studies have to be conducted in order to find methods which produce mechanically robust capsules. The main goal of this paper is to find a suitable method to produce robust capsules using different concentration of the chemicals at different pH and also implementing some modifications such as addition of cross-linkers in preparation procedure. In this paper comparison of three different encapsulation techniques were studied based on the mechanical robustness of the capsules. The three different techniques were calcium mineralized alginate-chitosan capsules, alginate capsules coated with 2% chitosan (2% AC) and genipin crosslinked alginate-chitosan (GCAC) capsules. The results indicate that GCAC capsules are most robust and were good enough for prolonged use since most of the capsules were not deformed in mechanical strength test. There were slight differences in the diameter and membrane thickness before and after swelling. No negative influence was observed on the yeast growth when applying the cross-linker. The results of this study will hopefully add valuable information and helps in further studies using other cross-linkers to prepare robust capsules. / Program: Industrial Biotechnology

O presente trabalho teve por objetivo avaliar condições de imobilização da levedura Scheffersomyces stipitis NRRL Y-7124 pelo método do aprisionamento em gel de alginato de cálcio visando à produção de bioetanol em biorreator STR tipo cesta à partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Primeiramente, realizou-se as etapas de obtenção, destoxificação e caracterização do hidrolisado hemicelulósico de bagaço de cana-de-açúcar. Realizou-se em seguida um screening objetivando a seleção de um meio de cultivo adequado para a produção de etanol por esta levedura. O meio escolhido foi aquele onde se suplementou o hidrolisado com extrato de levedura (3,0 g/L), peptona (5,0 g/L), (NH4)2SO4 (2,0 g/L) e CaCl2 (0,1 g/L), onde verificou-se um fator de conversão de xilose à etanol (Yp/s) de 0,33 g/g. As condições de imobilização da levedura foram então avaliadas por planejamento fatorial 23 completo onde os fatores concentração de alginato de sódio, concentração do cloreto de cálcio e tempo de cura foram investigados. Após a análise estatística, as condições 2% de alginato de sódio, 0,1M de cloreto de cálcio e tempo de cura de 12 horas foram fixadas para as etapas seguintes. Nestas condições, avaliou-se então a influência da concentração de células à serem imobilizadas e agitação durante a fermentação a partir de um planejamento fatorial 22 completo, definindo-se assim 10 g/L de células e 100 rpm como condições ideais. Após a determinação das condições de imobilização do processo, verificou-se a estabilidade das células imobilizadas em repetidos ciclos fermentativos, para isso cinco bateladas repetidas em frascos Erlenmeyer foram realizadas. Observou-se que apesar da levedura assimilar xilose e produzir etanol em todos os ensaios, uma diminuição na eficiência da fermentação foi verificada, diminuindo em 24% da terceira para a quarta batelada, indicando assim que a levedura imobilizada era viável para o sistema de batelada repetida em até 3 ciclos nas condições estudadas. Iniciou-se então ensaios fermentativos em biorreator STR tipo cesta, realizando-se ensaios em meio sintético e em hidrolisado hemicelulósico. Observou-se reprodutibilidade nos ensaios utilizando os diferentes meios, com um valor de Yp/s de 0,21g/g e uma produtividade volumétrica de 0,15 g/L.h em ambos os ensaios. Fermentações em sistema de bateladas repetidas foram realizadas neste biorreator STR tipo cesta. Realizou-se cinco ciclos consecutivos, ao final dos quais observou-se comportamento semelhante às bateladas repetidas realizadas em frascos Erlenmeyer, na qual a partir de três ciclos a capacidade fermentativa da levedura S. stipitis diminuiu, apresentando uma produtividade volumétrica em torno de 0,16 g/L.h nas três primeiras bateladas. O gel de alginato de cálcio apresentou considerável estabilidade em sistema de bateladas repetidas indicando a possibilidade de sua utilização nesse processo. Embora os resultados obtidos neste trabalho sejam inferiores aos observados com células livres por outros autores, os mesmos demonstraram o potencial do emprego do gel de alginato de cálcio e da levedura Scheffersomyces stipitis imobilizada para a produção de bioetanol a partir de bagaço de cana-de-açúcar e contribuíram para os conhecimentos sobre a fermentação de hidrolisado hemicelulósico à etanol. / This study aimed to evaluate immobilization conditions for the yeast Scheffersomyces stipitis NRRL Y-7124 entrapped in calcium alginate gel in basket type of STR bioreactor for ethanol production from sugarcane bagasse hemicellulosic hydrolysate. For this purpose, first the steps to obtain the hydrolysate by dilute acid pretreatment, detoxification and characterization of hydrolysate was performed. Then, a screening aiming the selection of a suitable culture medium suitable for ethanol production by this yeast was carried out. The medium which showed maximum ethanol production (Yp/s, 0.33 g/g) was selected to continue the further studies. It was composed by the hydrolyzate supplemented with yeast extract (3.0 g/L), peptone (5.0 g/L), (NH4)2SO4 (2.0 g/L), CaCl2 (0.1g/L). The immobilization conditions of the yeast were then evaluated through a 23 factorial design where the three process variables i.e. concentration of sodium alginate, concentration of calcium chloride and reaction time were investigated. After statistical analysis, the optimum set of conditions (2% of sodium alginate, 0.1 M of calcium chloride and a reaction time of 12 hrs) were set to perform the following steps of this study. Subsequently, the influence of the cell concentration for immobilization and agitation during fermentation were studied considering a factorial design 22. This study revealed that 10 g/L of cells and 100 rpm were the optimum conditions for ethanol production via immobilized systems. After determination of the conditions for immobilization procedure, the stability of the immobilized cells were evaluated by repeated fermentation cycles, for that five repeated batches were performed in Erlenmeyer flasks. It was observed that despite the yeast assimilates xylose and produces ethanol in all assays, a decrease in the efficiency of the fermentation was verified from the third batch, revealing the 65% efficiency in the second batch and 39% in the fourth batch. This behavior indicates that the immobilized yeast is viable for repeated batch system only up to 3 cycles under the employed conditions. Fermentation tests in basket type STR bioreactor were carried out using synthetic medium and hemicellulosic hydrolysate as carbon source. Reproducibility was observed in assays using the different medium with ethanol yield (Yp/s) of 0.21 g/g and a volumetric productivity of 0.15 g/L.h in both assays. Fermentation assay in repeated batch system were carried out in STR basket type bioreactor. Five consecutive fermentation cycles were performed which eventually showed the similar behavior with the repeated batches conducted in Erlenmeyer flasks. The fermentative efficiency of the yeast S. stipitis was considerably good up to three cycles with a volumetric productivity of 0.16 g/L.h followed by a concomitant down fall. The calcium alginate gel showed a considerable stability in the experiments, indicating the viability of its application in repeated batch system. Although the results of this work are inferior to that observed by other authors using free cells, the calcium alginate gel potential is evident and the yeast Scheffersomyces stipitis showed to be capable to produce ethanol in immobilized form, contributing with knowledge for second generation ethanol production from sugarcane bagasse hemicellulosic hydrolysate adopting biochemical platform.

Bioethanol is a promising renewable biofuel and wheat is currently the main candidate asthe feedstock for its production in the UK context. The quality of the numerous varieties ofwheat developed in the past by plant breeders has been well examined in terms of bread, biscuitand pasta producing industries. In general, the end-use quality determination of wheat in termsof alcohol yield is less investigated. This work focused on understanding and predicting thealcohol yield from wheat according to its physical, physicochemical and chemicalcharacteristics. The research ran alongside the GREEN Grain project and utilised its wheatsamples, which consist of a range of wheat varieties, agronomic regimes and growing sitesfrom four harvests years 2005-2008. The combined dataset consists of a diverse range ofchemical, physicochemical and physical characteristics of the GREEN Grain wheats. An initial multivariate analysis (PCA) indicated that the first principal component, whichexplains most of the variability of the wheat characteristics, is related with the classification ofwheat as hard or soft. High alcohol yielding wheats typically have high starch, mealiness andalbumin+globulin fraction, and also low protein, gliadin fraction and hardness. They also havelarger and more spherical kernels. Analysis of Variance (ANOVA) was applied in order to identify differences between thevarieties, the sites and the application or not of N fertiliser. The ANOVA showed that theapplication of N fertiliser increases all the protein components, although it increases the Gliadinand the LMW glutenins more. N fertiliser also yields smaller (TGW, width, depth) and moreelongated kernels. High alcohol yielding varieties tend to be softer with lower protein andlarger and more spherical kernels. This consistent variability allowed prediction of the alcoholyield based on easily measured parameters. The following model, based on the SKCS reportedvalues plus protein, could predict the alcohol yield with an R2 of about 78%:Alcohol yield = 466.62 - 5.07 × Protein - 0.21 × hardness + 11.6 × diameter ±6.94 l/dry tonIt is frequently hypothesised that larger and more rounded kernels produce more alcoholbecause they have a smaller relative amount of the unfermentable outer layers. In an effort totest this hypothesis, the pericarp thicknesses and the crease characteristics of the wheat sampleswere measured. It was found that pericarp thickness and crease dimensions vary with kernelsize, with significant differences between varieties. A physical model was developed thatconsiders these differences and calculates the endosperm to non-endosperm ratio. None of thevariables obtained by the physical model could be related to alcohol yield. The SKCS fundamental data were further analysed in an effort to improve the alcoholyield predictability. It was found that the averaged Crush Response Profiles are morereproducible than the hardness index itself. It was shown that the initial peak does not occurbecause of the "shell" (i.e. the bran layers) as suggested in the literature, but because of thecrease. Examination of the effects of moisture content on the aCRPs showed that their 1stquarter is equivalent to the stress-strain plots of dedicated rheological tests. The remaining partsof the curve relate to the post-failure behaviour of the kernels and with hardness as used incereal science. The aCRP parameters could improve the alcohol yield predictability of theGREEN Grain wheats to an R2 of about 82.3% and a standard error of the regression of6.3 l/dry ton. Further standardisation and calibration with respect to the moisture content and tothe size of the kernels could improve the predictions even further. Textural testing of cereals is constrained by the complexity of the wheat kernel structureand exacerbated by the between-kernel variation. The current work has demonstrated howSKCS data can be interpreted more insightfully in order to improve end-use quality predictions. The aCRP parameters clearly contain rheological information about wheats. Further research toestablish their examination by more standardised methodologies will allow effectiveinvestigation of connections between the rheological properties, chemical characteristics,processing behaviour and end-use quality prediction of wheat.